EP1490111A2 - Utilisation d'agonistes de recepteur edg pour le traitement de l'hypertension - Google Patents

Utilisation d'agonistes de recepteur edg pour le traitement de l'hypertension

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Publication number
EP1490111A2
EP1490111A2 EP02790454A EP02790454A EP1490111A2 EP 1490111 A2 EP1490111 A2 EP 1490111A2 EP 02790454 A EP02790454 A EP 02790454A EP 02790454 A EP02790454 A EP 02790454A EP 1490111 A2 EP1490111 A2 EP 1490111A2
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EP
European Patent Office
Prior art keywords
receptor
edg
encoded
lysohospholipid
substance
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP02790454A
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German (de)
English (en)
Inventor
Gerhard J. Molderings
Michael BRÜSS
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Abbott Products GmbH
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Solvay Pharmaceuticals GmbH
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Publication of EP1490111A2 publication Critical patent/EP1490111A2/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4168—1,3-Diazoles having a nitrogen attached in position 2, e.g. clonidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Definitions

  • the present invention investigates noradrenaline release-inhibiting receptors on PC12 cells devoid of ⁇ 2 - and CBT receptors, in particular with regard to similarities to presynaptic imidazoline and edg receptors.
  • the invention particularly pertains to a method of treatment of hypertension, said method involving the edg-receptor.
  • the receptor, membranes and cells comprising said receptor, and assays for screening compounds with said receptor are discussed in the context of the invention.
  • the presynaptic imidazoline receptor is blocked with low potency by the ⁇ 2 -adrenoceptor antagonist rauwolscine and with moderate potency by the CB-, cannabinoid receptor antagonists SR141716A and LY320135 (Molderings and G ⁇ thert, 1998, 1999, Molderings et al., 1999).
  • the presynaptic imidazoline receptor is an entity independent of ⁇ 2 -adrenoceptors and cannabinoid receptors
  • a cell line which expresses such release-inhibiting imidazoline receptors but not ⁇ 2 - and CB T cannabinoid receptors would be helpful.
  • the rat pheochromocytoma cell line PC12 was hypothesized to be suitable for this purpose because it possesses many properties of sympathetic neurones and adrenal chromaffin cells (for review, see Greene and Tischier, 1976).
  • the ⁇ 2 -adrenoceptor has been reported to be absent (Schwegler and Bonisch, 1986; Duzic and Lanier, 1992); however, this is not generally accepted since other groups proposed its existence (Gatti et al., 1988; Gollasch et al., 1992; Kim et al., 1993). Therefore, the first aim of the present study was to establish the absence or presence of ⁇ 2 - adrenoceptors and to examine whether PC12 cells lack CB ! cannabinoid receptors, using radioligand binding and molecular biological techniques.
  • the second aim was to investigate whether PC12 cells express imidazoline receptors with pharmacological properties comparable to those of the presynaptic imidazoline receptors on the sympathetic neurones of man, rat and rabbit. If so, the third aim finally was to investigate whether the release-inhibiting imidazoline receptor can be classified as a member of an already known receptor family defined by its pharmacological and/or molecular biological properties. The forth aim finally was to establish a novel method of
  • a method of treatment and/or prophylaxis of hypertension which involves the administering to a mammal a therapeutically active amount of an edg-receptor agonist or a pharmaceutically acceptable salt thereof.
  • the invention pertains to a method of treatment and/or prophylaxis of hypertension in which the edg-receptor agonist is a highly selective -receptor agonist and essentially devoid of ⁇ 2 -receptor agonist activity.
  • the method of treatment and/or prophylaxis of hypertension involves an edg-receptor agonist which has an imidazoline compound structure.
  • hypertension may be treated and/or prophylacted by administering imidazoline compounds showing highly selective agonist activity for the edg-receptor, however without causing ⁇ 2 -receptor induced side effects.
  • Veratridine-evoked [ 3 H]noradrenaline release from PC12 cells was inhibited by ⁇ molar concentrations of the imidazoline and guanidine derivatives cirazoline, clonidine, aganodine, 1 ,3-di(2-tolyl)guanidine, BDF6143 and agmatine, and of the cannabinoid receptor agonist WIN55.212-2 (R(+)-[2,3-dihydro-5-methyl-3- [(morpholinyl) methyl] pyrrolo- [1 ,2,3-de] -1 ,4-benzoxazin-yl] (l-naphthalenyl)-methanone mesylate) but not by noradrenaline.
  • the inhibitory effect of clonidine was antagonized by ⁇ molar concentrations of rauwolscine and SR141716A (N-[piperidin-1 -yl]-5-[4-chlorophenyl]-1-[2,4-dichlorophenyl]-4-methyl-1 H-pyrazole-3- carboxamide).
  • the potencies of the agonists and antagonists were compatible with an action at previously characterized presynaptic imidazoline receptors.
  • 1-Oleoyl-lysophosphatidic acid but not sphingosine-1 -phosphate produced an inhibition of release that was antagonized by 30 ⁇ M rauwolscine, 1 ⁇ M SR141716A and 10 ⁇ M LY320135 as well as by pretreatment of the cells with 100 ⁇ M clonidine for 72 h.
  • PC12 cells are not endowed with ⁇ 2 -adrenoceptors and CBi cannabinoid receptors, but with an inhibitory receptor recognizing imidazolines, guanidines and WIN55.212-2 similar to that on sympathetic nerves.
  • the PCR results and the ability of 1-oleoyl-LPA to mimic these drugs suggest that the release-inhibiting receptor may be an edg-encoded lysophospholipid receptor.
  • the invention therefore enables to a method of treatment of hypertension by involving the edg-receptor.
  • hypertension may be treated and/or prophylacted by administering compounds showing highly selective agonist activity however without causing ⁇ 2 -receptor induced side effects.
  • the invention also provides appropriate screening tools for identifying compounds acting with high selectivity as receptor agonists however lacking ⁇ 2 -receptor induced side effects.
  • the receptor, membranes and cells comprising said receptor, and assays for screening compounds with said receptor are discussed in the context of the invention.
  • the cells were preincubated with culture medium containing 10 nM [ 3 H]noradrenaline and, after 60-min exposure to [ 3 H]noradrenaline-free culture medium, incubated with [ 3 H]noradrenaline-free HEPES-buffered salt solution containing 15 mM KCI and 1 ⁇ M desipramine throughout.
  • Clonidine and the interacting drug under study (rauwolscine: 3 ⁇ M, (•); 30 ⁇ M, ( ⁇ ); SR141716A: 1 ⁇ M, solid column; 10 ⁇ M, ( ⁇ ); LY320135: 10 ⁇ M, dashed column) or its solvent ((o), open column) were present from the onset of incubation with the HEPES buffer until the end of the experiment; 5-min stimulation with 1 mM veratridine was carried out 60 min after onset of incubation with the buffer. Evoked [ 3 H]noradrenaline release was expressed as percentage of evoked release in control experiments carried out in parallel without clonidine.
  • CBi receptor agonists CP55.950, anandamide, WIN55.212-2 and WIN55.212-3
  • the cells were preincubated with culture medium containing 10 nM [ 3 H]noradrenaline and, after 60-min exposure to [ 3 H]noradrenaline-free culture medium, incubated with [ 3 H]noradrenaline-free HEPES-buffered salt solution containing 15 mM KCI, and 1 ⁇ M desipramine throughout.
  • the agonist and antagonist under study (3 ⁇ M rauwolscine, open columns; 1 ⁇ M SR141716A + 3 ⁇ M rauwolscine, solid column) were present from the onset of incubation with the HEPES buffer until the end of the experiment; 5-min stimulation with 1 mM veratridine was carried out 60 min after onset of incubation with the buffer. Evoked [ 3 H]noradrenaline release was expressed as percentage of veratridine-evoked release in control experiments carried out in parallel without CP55,950, anandamide, WIN55,212-2 or WIN55, 212-3. Means ⁇ SEM of 6 experiments in each group. * P ⁇ 0.05, ** P ⁇ 0.01 , * ** P ⁇ 0.001 (compared with the corresponding controls); + P ⁇ 0.05 (compared with the effect of 0.3 M CP55.940 in the presence of 3 M rauwolscine).
  • the cells were preincubated with culture medium containing 10 nM [ 3 H]noradrenaline and, after 60-min exposure to [ 3 H]noradrenaline-free culture medium, incubated with [ 3 H]noradrenaline-free HEPES-buffered salt solution containing 15 mM KCI and 1 ⁇ M desipramine throughout.
  • 1-Oleoyl- LPA and the interacting drug under study (rauwolscine: 30 ⁇ M, second solid column; SR141716A: 1 ⁇ M, third dashed column; pretreatment with 100 ⁇ M clonidine: fourth cross-hatched column)or its solvent ( ; first column) or 1-palmitoyl-LPA ( ) were present from the onset of incubation with the HEPES buffer until the end of the experiment; 5-min stimulation with 1 mM veratridine was carried out 60 min after onset of incubation with the buffer. Evoked [ 3 H]noradrenaline release was expressed as percentage of evoked release in control experiments carried out in parallel without the lysophosphatidic acid derivatives.
  • a method of treatment and/or prophylaxis of hypertension which involves the administering to a mammal a therapeutically active amount of an edg-receptor agonist or a pharmaceutically acceptable salt thereof.
  • the invention pertains to a method of treatment and/or prophylaxis of hypertension in which the edg-receptor agonist is a highly selective 1,-receptor agonist and essentially devoid of ⁇ 2 -receptor agonist activity.
  • the method of treatment and/or prophylaxis of hypertension involves an edg-receptor agonist which has an imidazoline compound structure.
  • hypertension may be treated and/or prophylacted by administering imidazoline compounds showing highly selective h agonist activity for the edg-receptor, however without causing ⁇ 2 -receptor induced side effects.
  • the edg- receptor agonist or its physiologically compatible salt e.g. acid-addition salts
  • any suitable route of administration may be employed, i.e. the compound can be administered orally, intravenously or transdermally in conventional pharmaceutical preparations.
  • the therapeutically active quantities of the compounds that are used for the treatment and/or prophylaxis of hypertension can be contained together with customary pharmaceutical excipients and/or additives in solid or liquid pharmaceutical formulations.
  • solid dosage forms are tablets, coated tablets, capsules, powders, granules or suppositories. These solid dosage forms can contain standard pharmaceutical inorganic and/or organic excipients such as lactose, talc or starch in addition to customary pharmaceutical additives such as lubricants or tablet disintegrants.
  • Liquid preparations such as solutions (i.e.
  • solutions to be administered iv) suspensions or emulsions of the active ingredients can contain the usual diluents such as water, oil and/or suspending aids such as polyethylene glycols and the like. Further additives such as preservatives and the like may also be added.
  • the active ingredients can be mixed and formulated with the pharmaceutical excipients and/or additives in a known manner.
  • the active ingredients may be mixed with the excipients and/or additives in the usual manner and granulated in a wet or dry process. Granules or powder can be filled directly into capsules or compressed into tablet cores. If desired, these can be coated in a known manner.
  • the active compounds are dissolved in a suitable liquid carrier and optionally suitable adjuvants may be added.
  • compositions suitable for injection may be sterilised solutions containing a therapeutically active amount of an edg-receptor agonist or its physiologically compatible salt dissolved in a physiologically acceptable isotonic saline solution (i.e., containing 0.9 % by wt. sodium chloride).
  • a physiologically acceptable isotonic saline solution i.e., containing 0.9 % by wt. sodium chloride
  • PC12 cells which represent a neuronal-like rat pheochromocytoma cell line
  • release-inhibiting receptors that resemble previously characterized presynaptic imidazoline receptors on sympathetic nerves (Molderings and G ⁇ thert 1999), but lack ⁇ 2 -adrenoceptors and cannabinoid CBi receptors.
  • the ultimate aim was to investigate whether the release-inhibiting receptors can be classified as belonging to a receptor family which has already been defined by its pharmacological and molecular properties.
  • veratridine-evoked tritium overflow from PC12 cells preincubated with [ 3 H]noradrenaline was determined, which under the present conditions (blockade of the neuronal noradrenaline transporter) reflects release of tritiated and endogenous noradrenaline from the PC12 cells (for details, see Bonisch et al. 1990).
  • Veratridine stimulates Na + influx mainly through voltage-dependent Na + channels (Ulbricht, 1998).
  • cannabinoid receptor antagonists SR141716A (Rinaldi-Carmona et al., 1995) and LY320135 (Felder et al., 1998) counteracted the inhibitory effect of clonidine but with potency lower than at CB T receptors.
  • the cannabinoid receptor agonist WIN55.212-2 also produced a low potency inhibition of [ 3 H]noradrenaline release, whereas WIN55.212-3, an enantiomer which is inactive at CBi cannabinoid receptors but shares the lipophilicity of WIN55,212-2, did not.
  • the cannabinoid receptor agonist CP55.940 at a concentration of 0.3 ⁇ M also inhibited [ 3 H]noradrenaline release, an effect which was antagonized by 1 ⁇ M SR141716A. The more the concentration of CP55.940 was increased above 0.3 ⁇ M, the more the inhibitory effect disappeared and was finally reversed to a facilitation.
  • the receptors mediating inhibition of [ 3 H]noradrenaline release in cardiovascular tissue and PC12 cells resemble each other with respect to their pharmacological properties and share properties with the ⁇ 2 -adrenoceptor and the CB ! cannabinoid receptor; however, they are not identical with those receptors because the expression of ⁇ 2 - and CBi cannabinoid receptors on PC12 cells was ruled out by our radioligand binding and PCR experiments.
  • 1-oleoyl-LPA The ability of 1-oleoyl-LPA to mimic the pharmacological properties of the imidazoline and guanidine derivatives at the release-inhibiting receptor points to the possibility that this receptor may represent a lysophospholipid receptor.
  • lysophospholipid receptor types At present eight lysophospholipid receptor types have been identified and cloned. They are provisionally termed edg1-8 according to the endothelial differentiation genes 1-8 which code for these receptors.
  • 1-Oleoyl-LPA is an agonist at edg2, edg4 and edg7, whereas it is almost not active at edgl , edg3, edg5, edg6 and edg8.
  • S1 P is an agonist at the latter receptors, whereas it is inactive at the former ones.
  • S1 P is an agonist at the latter receptors, whereas it is inactive at the former ones.
  • cDNA coding for edgl , edg4, edg6 and edg8 receptors was detectable in PC12 cells.
  • these results of the PCR experiments together with those of our release experiments and with the pattern of edg receptors that are activated by 1-oleoyl-LPA are at least compatible with the possibility that the release-inhibiting receptor may be identical with the edg2 or the edg7 receptor or a so far unknown lysophospholipid receptor at which 1-oleoyl-LPA acts as an agonist.
  • 1-oleoyl-LPA may also act via such a so-called imidazoline receptor.
  • PC12 cells were found to express mRNA for 1-oleoyl-LPA-activated edg2 and edg7 receptors.
  • these results are compatible with the possibility that the release-inhibiting receptors so far known as presynaptic non-l ⁇ /non-l 2 - imidazoline receptors are edg-encoded lysophospholipid receptors. More direct support for this conclusion can be given by experiments on cells transfected with cDNAs coding for edg receptors.
  • the invention may also be used to screen for compounds for the treatment of hypertension.
  • the invention also pertains to a method for determining or identifying whether a substance, preferably a candidate compound, is a potential ligand of an edg-encoded lysohospholipid receptor, preferably of a mammalian edg-encoded lysohospholipid receptor, wherein said method comprises: a. contacting cells expressing the edg-encoded lysohospholipid receptor polypeptide, or contacting a receptor membrane preparation comprising said edg-encoded lysohospholipid receptor polypeptide, with a labeled ligand, such as described in the specification, in the presence and in the absence of the substance; and b. measuring the binding of the labeled ligand to the edg-encoded lysohospholipid receptor.
  • the invention also pertains to a method for determining or identifying whether a substance, preferably a candidate compound, modulates the interaction of ligand, such as described in the specification, with edg-encoded lysohospholipid receptor polypeptide, preferably with a mammalian edg-encoded lysohospholipid receptor polypeptide, wherein said method comprises: a.
  • contacting cells expressing on the surface thereof an edg-encoded lysohospholipid receptor polypeptide said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor, with a substance, preferably with a candidate compound, to be screened under conditions to permit binding to the receptor; and b. determining whether the substance, preferably the candidate compound, modulates the interaction a ligand, such as described in the specification, and the edg-encoded lysohospholipid receptor polypeptide by detecting an increase or decrease in the signal normally generated by the interaction of the ligand, such as described in the specification, with the receptor after interaction of the ligand with the receptor.
  • a ligand such as described in the specification
  • the invention also pertains to a method for determining or identifying whether a substance, preferably a candidate compound, inhibits or antagonizes the interaction of a ligand, such as described in the specification, with an edg-encoded lysohospholipid receptor polypeptide, preferably with a mammalian edg-encoded lysohospholipid receptor polypeptide, wherein said method comprises: a.
  • contacting cells expressing on the surface thereof an edg-encoded lysohospholipid receptor polypeptide said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor, with a substance, preferably with a candidate compound, to be screened under conditions to permit binding to the receptor; and b. determining whether the substance, preferably the candidate compound, inhibits or antagonizes the interaction of the ligand, such as described in the specification, and the edg-encoded lysohospholipid receptor polypeptide by detecting an increase or decrease in the signal normally generated by the interaction of the ligand, such as described in the specification, with the receptor after interaction of the ligand with the receptor.
  • the invention pertains to a method for determining or identifying whether a substance, preferably a candidate compound, is an agonist to an edg-encoded lysohospholipid receptor polypeptide, preferably to a mammalian edg-encoded lysohospholipid receptor polypeptide, wherein said method comprises: a. contacting cells expressing on the surface thereof an edg-encoded lysohospholipid receptor polypeptide with a substance, preferably a candidate compound; and b.
  • the substance preferably the candidate compound, effects a signal generated by activation of the edg-encoded lysohospholipid receptor polypeptide, using a ligand, such as described in the specification, as a positive control for the generation of a signal.
  • the invention also pertains to a method for determining or identifying whether a substance, preferably a candidate compound, is a modulator, an agonist or antagonist to a noradrenaline release-inhibiting edg-encoded lysohospholipid receptor polypeptide, preferably to a mammalian edg-encoded lysohospholipid receptor polypeptide, wherein said method comprises: a. loading cells expressing on the surface thereof an edg-encoded lysohospholipid receptor polypeptide with labelled noradrenaline, then b.
  • the invention pertains to a screening method as described before, wherein the substance, preferably the candidate compound, is effective with regard to dysfunctions, disorders or diseases of the cardiovascular system, including the heart, of the nervous system, including the central nervous system, and also of glucose and insulin metabolism or with regard to dysfunctions, disorders or diseases related to increased sympathetic tonicity.
  • the substance, more preferably the candidate compound is effective with regard to dysfunctions, disorders or diseases associated with the cardiovascular system, preferably including blood pressure control, e.g. hypertension or vasodilatation, myocardial ischaemia, ischaemic preconditioning, cardioprotective activity, or other heart related diseases.
  • isolated rat pheochromocytoma PC12 cells comprising an edg-encoded lysohospholipid receptor may be used.
  • the isolated rat pheochromocytoma PC12 cells may be used comprising an edg-encoded receptor which is a nordrenaline release-inhibiting receptor.
  • isolated rat pheochromocytoma PC12 cells are used comprising a receptor which is encoded by the genes edg2, edg3, edg5 and /or edg7.
  • the isolated rat pheochromocytoma PC12 cells preferably are devoid of ⁇ 2 -receptors and/or CB ! -receptors.
  • the edg receptor is not only binding imidazoline compounds but that after binding the edg receptor responds with activation.
  • the edg receptor may be used for activation by imidazoline compounds e.g. for the treatment and/or prophylaxis of hypertension or optionally also for the treatment and/or prophylaxis of other dysfunctions, disorders or diseases of the cardiovascular system, e.g. as those stated supra.
  • imidazoline compounds may be provided that are highly selective with essentially reduced or even no ⁇ 2 -indiuced side effects. This provides a substantial improvement over the state of the art, e.g. it is possible to avoid ⁇ 2 -induced side effects like sedation as known from the ⁇ 2 -/lrCompound clonidine.
  • PC12 cells were cultured in suspension culture as described by Harder and Bonisch (1984).
  • the culture medium was composed of 85% RPMI 1640 medium (Gibco, Düsseldorf, FRG), 10% heat-inactivated horse serum (Gibco), 5% fetal calf serum (Gibco) and was buffered with 24 mM NaHC0 3 .
  • PC12 cells were cultured in a humidified C0 2 -incubator (at 37°C and in the presence of 9% C0 2 ) on dishes (60 mm, Nunc) coated with polyomithine 0.1 g/l (in 0.15 M boric acid, and 67 mM NaOH, adjusted with HCl to pH 8.4).
  • Each culture dish contained about 10 7 cells corresponding to about 1 mg cell protein.
  • Undifferentiated PC12 cells were harvested and homogenized in HEPES buffer ( HEPES-Na + 5 mM, EGTA 0.5 mM, MgCI 2 0.5 mM, ascorbic acid 0.1 mM, pH 7.4, 4°C) with a polytron homogenizer for 1 min and then centrifuged for 10 min (1000xg; 4°C) to pellet cellular debris. The supernatant was centrifuged for 30 min (40000xg; 4°C) and the pellet resuspendend in fresh HEPES buffer and washed twice. Finally, the washed pellet was stored at -80°C until use.
  • HEPES buffer HEPES-Na + 5 mM, EGTA 0.5 mM, MgCI 2 0.5 mM, ascorbic acid 0.1 mM, pH 7.4, 4°C
  • the membranes were centrifuged (20 min, 40000g, 4°C), resuspended in the buffer, homogenized by ultrasonication and diluted to a final protein concentration of about 0.6 mg/ml.
  • a 400- ⁇ l aliquot of the membranes was incubated for 30 min with 16.8 nM [ 3 H]rauwolscine or for 60 min with 10 nM [ 3 H]SR141716A at room temperature in a final volume of 0.5 ml.
  • the reaction was stopped by rapid vacuum filtration with a Brandel cell harvester through Whatman GF/C glass-fiber filters presoaked with polyethylenimine 0.5 M followed by rapid washing of the incubation tubes and filters with 10 ml ice-cold buffer. Filters were placed in 6 ml of scintillation fluid and shaken overnight, and the radioactivity was determined by liquid scintillation counting at 44% efficiency.
  • Non-specific binding was defined as [ 3 H]rauwolscine binding in the presence of 10 ⁇ M rauwolscine and as [ 3 H]SR141716A binding in the presence of 3 ⁇ M CP55.940. Results are expressed as mean values ⁇ SEM. All experiments were carried out in triplicate. Data were analyzed using the least-squares fitting program PRISM (GraphPad Software Inc., San Diego, USA).
  • Template DNAs were first strand cDNA from undifferentiated PC12 cells and genomic DNA prepared from rat whole blood. To prepare cDNA, cells were chemically dissected; total RNA from PC12 cells was isolated with the Quiagen RNeasy kit according to the manufacturers protocol. Then oligo dT primed first strand cDNA synthesis was performed with AMV Reverse Transcriptase (Promega), followed by RNase digestion, phenol/chloroform extraction and cDNA purification with Chroma Spin 30 columns (Clontech). Genomic DNA was purified from rat whole blood with the Quiagen QIAamp Blood Midi kit according to the manufactorer ' s protocol.
  • the purified DNA was used as template for subsequent PCR amplification of the receptor DNA under the following conditions: primer sequences for the rat ⁇ 2A -adrenoceptor [sense primer: 5 ' - catctccttcccgccactcatc-3 ' ; antisense primer: 5 ' -atacgcacgtagaccaggatc-3 ' ] and for the rat CBT cannabinoid receptor [sense primer: 5 ' -ctggc(ac)(gt)(ag)gc(agct)gac(agct)tcctg-3 ' ; antisense primer: 5-a(gt)(ag)g(ct)(ag)tagat(agct)a(agct) (agct)gggttc-3 ' ] were chosen according to the sequences of Gene Bank ace.
  • Primer sequences for the lysophospholipid receptors were chosen according to the following sequences: edgl : ace. no. U10303 (sense primer: 5 ' -cttcagcctccttgctatcg-3 ' ; antisense primer: 5 ' -gcaggcaatgaagacactca- 3 ' ); edg2: ace. no.
  • AF014418 (sense primer: 5 ' -ccaaacta cagcactctcatg-3 ' ; antisense primer: 5 ' - gcttccttctaaaccacagag-3 ' ); edg3: ace. no. NM005226 (sense primer: 5 ' -tcagggagggcagtatgttc-3 ' ; antisense primer: 5 ' -ctgactttcgaagaggatgg-3 ' ); edg4: ace. no.
  • AW141943 (sense primer: 5 ' -ggagtacctgcgcggcatg-3 ' ; antisense primer: 5 ' - catggcctcggacatggacac-3 ' ); edg7: ace. no.
  • AW107032 (sense primer: 5 ' -atgaatgagtgtcactatgac-3 ' ; antisense primer: 5 ' -catacatgtagatgcgtacgt-3 ' ); edg ⁇ : ace. no.
  • PCR was performed in a total volume of 100 ⁇ l containing 15 nM primer (each), 5 U Taq DNA Polymerase (Gibco), 2 mM MgCI 2 , 200 ⁇ M dNTPs (each), 10 ⁇ l 10xTaq-Buffer (Gibco) and 3-5 ⁇ l template DNA. PCR was performed for 37-40 cycles.
  • PCR products were separated by agarose gel electrophoresis, and the band of interest was cut off the gel, purified with GeneElute" columns (Supelco), ligated into the TA-cloning" vector pCR2.1 (Invitrogen) and transformed into E.coli InV * (Invitrogen).
  • the subcloned DNA fragments were sequenced with an automated sequencer (Li-COR 4200, MWG- Biotech, Ebersberg, Germany) and the Thermo Sequenase fluorescent labelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham, Freiburg, Germany).
  • the cell dishes were transferred to a water bath of 37° C (onset of the experiment) and the cells were then incubated until the end of the experiment with prewarmed (37° C) [ 3 H]noradrenaline-free HEPES-buffered salt solution (composition in mM unless stated otherwise: NaCI 125, KCI 15, KH 2 P0 4 1.2, CaCI 2 2.6, MgS0 4 1.2, HEPES-NaOH (pH 7.4) 25, D(+)-glucose 5.6, L(+)-ascorbic acid 1.0) with a change of the medium every 5 min (total incubation time in the HEPES buffer: 85 min).
  • [ 3 H]noradrenaline-free HEPES-buffered salt solution composition in mM unless stated otherwise: NaCI 125, KCI 15, KH 2 P0 4 1.2, CaCI 2 2.6, MgS0 4 1.2, HEPES-NaOH (pH 7.4) 25, D(+)-
  • the solution contained 1 g/1 bovine serum albumin and 1 ⁇ M desipramine, respectively (Friedrich and Bonisch, 1986).
  • the agonist and/or antagonist under study was added to the HEPES buffer at the onset of the exposure to this buffer. Sixty min after onset of incubation with the buffer 1 mM veratridine was added for 5 min.
  • the HEPES-buffered salt solution contained 4.8 instead of 15 mM KCI and stimulation was carried out either by 1 mM veratridine or by increasing the KCI concentration to 15 mM (time schedule identical to the standard procedure).
  • culture medium was changed daily (instead of changing it every 3 days as standard condition) and 100 ⁇ M clonidine was added to the medium for 72 h.
  • the cells were then washed twice with clonidine-free culture medium (with a change of the medium after 30 min). Subsequently the cell dishes were transferred to a water bath of 37° C (onset of the experiment) and the cells were then incubated until the end of the experiment with prewarmed (37° C) [ 3 H]noradrenaline-free HEPES-buffered salt solution and veratridine was administered 2 h after the end of exposure to clonidine. Basal efflux of tritium was determined in the 5-min period before and in that 20 min after onset of stimulation.
  • Veratridine-evoked tritium overflow was determined in the four 5-min periods after the onset of veratridine application. At the end of the experiment, the cells were solubilized by 0.1% v/v TritonX-100 (in 5 mM Tris-HCI, pH 7.4). The radioactivity of the solubilized cells und of the wash-out samples was determined by liquid scintillation counting.
  • Rauwolscine hydrochloride agmatine sulfate, noradrenaline base, 1-oleoyl-lysophosphatidic acid, sphingosine-1 -phosphate (Sigma, Kunststoff, FRG); cirazoline hydrochloride (Synthelabo, Paris, France); aganodine, moxonidine, 4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline hydrochloride (BDF 6143; Beiersdorf, Hamburg, FRG); desipramine hydrochloride (Ciba-Geigy, Wehr, Germany); clonidine hydrochloride (Boehringer, Ingelheim, FRG); N-[piperidin-1-yl]-5-[4-chlorophenyl]-1- [2,4-dichlorophenyl]-4-methyl-1 H-pyrazole-3-carboxamide (SR141716A; Sanofi, Montpellier, France); [
  • Drugs were dissolved in saline with the following exceptions: DTG was dissolved in methanol; SR141716A, LY320135, anandamide, WIN55.212-2, WIN55.212-3, CP55.940, 1-oleoyl-LPA, 1- palmitoyl-LPA and S1 P were dissolved in dimethylsulfoxide (final maximum concentration in the buffer solution 1%). The stock solutions were further diluted in the buffer. Corresponding control experiments were run with the solvent only.
  • veratridine stimulus in the presence of 15 mM KCI was applied as the standard stimulation procedure with this depolarizing alkaloid.
  • the veratridine-evoked tritium overflow reflects exocytotic release of tritiated and unlabelled noradrenaline (for details, see Discussion) and is denoted as [ 3 H]noradrenaline release in the following text, table and figures.
  • the basal tritium efflux from the cells incubated with the buffer containing 15 mM KCI during the 5-min period before stimulation with veratridine amounted to 1.31 ⁇ 0.10 % of the tritium present in the cells before this collection period corresponding to 593 ⁇ 71 dpm/well (n 24 in a representative series of control experiments). It was not altered by the antagonists or agonists investigated in this study.
  • clonidine In the absence of rauwolscine, clonidine also concentration-dependently inhibited veratridine- evoked [ 3 H]noradrenaline release (Fig. 2A, open circles).
  • the concentration-response curve of clonidine in the presence of 3 ⁇ M rauwolscine did not substantially differ from that in the absence of rauwolscine (Fig. 2A, closed circles). However, this curve was shifted to the right by 30 ⁇ M rauwolscine (Fig. 2A, closed triangles) and SR141716A (Fig.
  • Primer pairs suitable to identify the cDNAs for the rat 2A and CBi cannabinoid receptors were applied in PCR experiments.
  • fragments were amplified by PCR from the genomic DNA from rat blood with the same primer pairs. Sequencing of these fragments revealed that they code for the r 2A -adrenoceptor and the rCBi receptor.
  • no message for the r 2A and the rCB ! receptors were found with the respective primer pairs in the cDNA from the undifferentiated PC12 cells (results not shown).
  • Genomic DNA prepared from rat blood was also used to prove whether the chosen primer pairs for lysophospholipid receptors were suitable to identify the corresponding edg messages. After PCR amplification of this genomic DNA, PCR products were found that were identified by sequencing as partial sequences of edgl , edg2, edg3, edg4, edg5, edg6, edg7 and edg ⁇ (results not shown).
  • PCR amplification of PC12 cell cDNA using the same primer pairs resulted in PCR products coding for partial sequences of edg2, edg3, edg5 and edg7 but not of edgl , edg4, edg6 and edg8 (results not shown).
  • the cells were preincubated with culture medium containing 10 nM [ Hjnoradrenaline and, after 60-min exposure to [ 3 H]noradrenaline-free culture medium, incubated with [ 3 H]noradrenaline-free HEPES-buffered salt solution containing 15 mM KCI, 1 ⁇ M desipramine and 3 ⁇ M rauwolscine throughout.
  • Unlabelled noradrenaline or the imidazoline or guanidine derivatives under study was present from the onset of incubation with the HEPES buffer until the end of the experiment; 5-min stimulation with 1 mM veratridine was carried out 60 min after onset of incubation with the buffer.
  • RH7777 cells rat hepatoma cells stably transfected with cDNA encoding the h(edg2) receptor and PC12 cells were homogenized in ice-cold 20 mM HEPES buffer containing 10n mM EDTA (pH 7.4 at RT) with an ultraturrax homgenizer and centrifuged at 48,000 g, 4°C for 15 min. The pellet was resuspended in ice-cold 20 mM HEPES buffer containing 0.1 mM EDTA (pH 7.4 at RT) and recentrifuged at 48,000 g, 4° C for 15 min. The final pellet was resuspended in 20 mM HEPES buffer (pH 7.4 at RT).
  • Membranes were incubated in 0.5 ml of GTP-binding buffer containing GDP 0.1 ⁇ M, rauwolscine 10 ⁇ M (in the experiments with membranes from RH7777 cells), [ 35 S]GTP ⁇ S 0.05 nM and indicated test compound (LPA, clonidine or moxonidine diluted in assay buffer containing 1% fatty acid free BSA) for 45 min at 30° C.
  • the incubation was terminated by filtering over GF/B filters with a cell harvester, and the filters were washed once with 5 ml of ice-cold assay buffer. Radioactivity was counted by liquid scintillation spectrometry at an efficiency of > 95%.
  • LY320135 a novel cannabinoid CBi receptor antagonist, unmasks coupling of the CBi receptor to stimulation of cAMP accumulation. J. Pharmacol. Exp. Ther. 284, 291-297.
  • Nerve growth factor increases the number of functional Na channels and induces TTX-resistant Na channels in PC12 pheochromocytoma cells. J. Neurosci. 7, 1613-1625.

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Abstract

L'invention concerne une méthode de traitement et/ou de prophylaxie de l'hypertension consistant à administrer une dose thérapeutiquement active d'un agoniste de récepteur edg ou un sel pharmaceutiquement acceptable de celui-ci. De préférence, cet agoniste de récepteur edg est un agoniste de récepteur l1 hautement sélectif, et essentiellement dépourvu d'une activité d'agoniste de récepteur α2, et présente une structure d'imidazoline. Ainsi, l'hypertension peut être traitée sans provoquer d'effets secondaires induits par un récepteur α2. Cette invention concerne également des outils de criblage appropriés permettant d'identifier des composés agissant avec une grande sélectivité en tant qu'agonistes de récepteur l1, et ne présentant cependant pas des effets secondaires induits par le récepteur α2. En outre, l'invention concerne le récepteur, les membranes et les cellules comprenant ce récepteur, et les essais permettant de cribler les composés comportant ledit récepteur.
EP02790454A 2001-11-30 2002-11-28 Utilisation d'agonistes de recepteur edg pour le traitement de l'hypertension Withdrawn EP1490111A2 (fr)

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JP2006505248A (ja) 2002-06-07 2006-02-16 イーエス・セル・インターナショナル・プライヴェート・リミテッド 幹細胞における分化を制御する方法
AU2003301991A1 (en) * 2002-11-13 2004-06-03 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with human endothelial differentiation, sphingolipid g-protein-coupled receptor 5 (edg5)

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WO1999019513A2 (fr) * 1997-10-10 1999-04-22 Lxr Biotechnology, Inc. Procedes pour la detection de composes modulant l'activite d'un recepteur d'acide lysophosphatidique (lpa)
WO1999046277A1 (fr) * 1998-03-09 1999-09-16 Smithkline Beecham Corporation POLYNUCLEOTIDES ET POLYPEPTIDES D'EDG-1c HUMAIN ET LEURS PROCEDES D'UTILISATION
WO2000060056A1 (fr) * 1999-04-05 2000-10-12 Smithkline Beecham Corporation Gene edg5 de la souris
WO2001011022A1 (fr) * 1999-08-05 2001-02-15 Smithkline Beecham Corporation Recepteur edg3 couple a la proteine g des souris

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